The purpose of our research is to decipher the molecular mechanisms that allow the bacterium Legionella pneumophila to infect human cells and cause a severe pneumonia known as Legionnaires disease. Since L. pneumophila is ubiquitously found in freshwater habitats such as water fountains, air conditioning systems, or shower heads and faucets, the American population is frequently exposed to this organism. When inhaled by elderly or immunocompromised individuals, L. pneumophila can infect alveolar macrophages resulting in a potentially fatal respiratory infection. According to the Center for Disease Control and Prevention (CDC), the number of diagnosed Legionnaires' disease cases has doubled over the past decade, explaining why this disease is an emerging public health threat. The ability of L. pneumophla to establish a replication vacuole within infected cells is key to its virulence, yet the details of this process are not very well characterized. Over the past funding period, we have continued our study of the intracellular replication cycle of L. pneumophila in order to determine which bacterial molecules contribute to disease development, what their functions are, and how we can interfere with their activity. We have discovered that L. pneumophila uses proteins, so called effectors, that are injected into infected human cells where they manipulate the activity of human signaling proteins such as GTPases. By doing so, the bacterium takes control of the infected cell and reprograms it in a way that the host cell now supports bacterial replication instead of fighting the invading microbe. Remarkably, some of the effectors that are used by L. pneumophila to manipulate our cells have been been acquired during evolution from the host cell itself and are now being used by the bacterium against the host. For instance, we and others discovered that L. pneumophila has a set of effector proteins that attach or remove a post-translational modification to human GTPase in order to control its activity. In collaboration with a group in Spain, we successfully solved the three-dimensional structure of one of these effectors which provided important insight into how this bacterial protein functions at a molecular level. These data now provide a framework for the development of small molecule inhibitors that block the enzymes activity and, thus, its ability to control signaling proteins in human cells. Our work not only yields much-needed insight into the virulence strategies of L. pneumophila and related pathogens, but it also provides important clues about the delicately balanced signaling networks within our own cells and how they contribute to or counteract bacterial infections.